Journal: Nucleic Acids Research
Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
Figure Lengend Snippet: Flowchart of Recombination-based ‘Library vs Library’ Y2H system (RLL-Y2H) screening. ( A ) The yeasts with bait and prey plasmids are mated and only the yeasts containing interacting bait-prey pairs can grow on SD/-AHLT selection plates. The bait and prey plasmids are integrated into a single plasmid so that the interacting bait and prey genes are paired in a single DNA fragment. ( B ) The integrase PhiC31 recognition sites ATTB and ATTP are inserted right behind the bait and prey genes, respectively. PhiC31 is inserted into the backbone and is driven by the Pol III promoter. The integrase PhiC31 recognizes ATTB and ATTP sites and integrates the bait and prey plasmids into a big plasmid in vivo which unambiguously couples the interacting pair of specific bait and prey genes. The ADH1 promoters and polyadenylation sites, as well as their positions after the recombination were indicated by the green bars with letter ‘P’ and ‘T’, respectively. The stop codons are indicated by the red lines right behind Bait and Prey. ( C ) The recombinated bait and prey fragments are amplified and digested by Type II restriction enzyme MmeI to generate ∼110 bp fragments containing bait tail-ATTL-prey tail, followed by DNA sequencing library construction and high-throughput sequencing.
Article Snippet: To this end, type II restriction enzyme MmeI (which cuts DNA ∼20 bp downstream of its recognition site) recognition sequences were inserted right after bait and prey genes, respectively, to generate a∼110 bp DNA fragments containing a pair of 20 bp interacting proteins cDNA strands and ATTL linker in between (Figure ).
Techniques: Selection, Plasmid Preparation, In Vivo, Amplification, DNA Sequencing, Next-Generation Sequencing